Abstract

In humans, parturition is currently viewed as an intrauterine outbreak of inflammation, accompanied by a massive release of proinflammatory cytokines at the maternal–fetal interface that comprises the maternal decidua, placenta, and fetal membranes. At term, fetal membranes overlying the cervix, the future site of rupture, show altered morphology and are termed the zone of altered morphology (ZAM). These alterations occur in normal fetal membranes during late pregnancy, in preparation for labor. In this study, transcriptome, flow cytometry, electron microscopy, and immunohistochemistry analyses collectively highlight a local shift in gene expression and lymphocyte activation in the ZAM. Just before labor, we show that highly polymorphic HLA-A, -B, and -C determinants of fetal origin are selectively exposed in the ZAM to the maternal immune system. A graft rejection-like program occurs in the ZAM, which involves 1) the activation of cytotoxic decidual NK cells, and 2) the decline of decidual immunotolerant M2-like macrophages. Comparison with a prior cohort of fetal membranes shows that acute inflammation only takes place after these first steps of immune modifications. Our results therefore strongly argue in favor of local immune remodeling at the onset of parturition.

Introduction

The mechanisms underlying the onset of parturition remain poorly understood in humans (1–3). Gestation in singleton pregnancies lasts an average of 40 wk (280 d) from the first day of the last menstrual period to the date of delivery. Parturition involves phenotypic changes, including cervical remodeling and dilatation, uterine contractions, and fetal membrane rupture that together allow the expulsion of the fetus. A few weeks before delivery, gestational connective tissue becomes increasingly compliant, leading to the development of the lower uterine segment, cervical ripening, and focal remodeling of the fetal membranes (amnion and chorion) (4). Notably, at full-term pregnancy (>39 wk of gestation) (5), fetal membranes overlying the cervix present physical weaknesses, accompanied by the swelling and disruption of connective tissue; this zone was termed the zone of altered morphology (ZAM) (6, 7), whereas other regions located far from the cervix remain unaltered, which we termed the zone of intact morphology (ZIM) (see Fig. 1A). Little is known about the biochemical cascade causing these alterations.

A shift from an anti-inflammatory to a proinflammatory pathway is currently thought to have a pivotal role in parturition, based on the observations of the abundance of inflammatory cytokines and chemokines, the high activity of proteases, and degradation of extracellular matrix (ECM) in gestational tissues sampled just after vaginal delivery (3). Global uterine transcriptome comparisons of myometrial changes between human samples at the time of labor and mouse parturition models confirm the dominant role for inflammatory pathways in human labor (8). Inflammation impairs the function of the progesterone receptor and promotes the expression of contraction-associated genes in the myometrium (9), enhances estrogen and progesterone metabolism in the cervix, creating a microenvironment favorable for cervical ripening and dilatation (10), and induces fetal membranes weakening (11). However, in humans it is still unclear how maternal, fetal, or placental signals trigger these inflammatory events in the absence of microbial invasion or intrauterine infection.

The ZAM is characterized by a significant reduction of the thickness of the chorionic cellular layer (7). This cellular layer, composed mainly of trophoblasts, is in close association with the decidua parietalis, the uterine mucosa during pregnancy. This mucosa is made up of endometrial stromal cells and 40% leukocytes. Among these leukocytes, in early pregnancy, 70% are decidual NK (dNK) cells that regulate trophoblast invasion (12, 13), 20% are resident macrophages, participating in tissue homeostasis and immune balance (14), and 10% are T cells, acting in the maternal–fetal tolerance, with most being CD8+ effective memory T cells (15) and 15–25% of CD4+ cells being CD4+CD25+ regulatory T cells (Tregs) (16, 17). Distribution of the decidual leukocyte population varies with gestational age, with the percentage of dNK cells decreasing to 50% and T cells reaching 60–80% of decidual leukocytes at full-term pregnancy (18).

In this study, we demonstrate a remarkable shift in gene expression and lymphocyte activation at full-term pregnancy limited in the ZAM. Indeed, we show that at full-term gestation and before labor, the ZAM is characterized by immune activation and graft rejection molecular signatures. In this specific region, dNK cells become activated, and primarily M2-like resident macrophages sharply decline. This is concomitant with a switch in HLA Ag presentation at the trophoblast membrane surface, in which poorly polymorphic HLA-F molecules are replaced with classical, highly polymorphic HLA-A, -B, and -C molecules. Collectively, our findings reveal a preparturition unmasking of trophoblasts as non-self entities, associated with the activation of a graft rejection-like program in the ZAM at term before labor. These immune modifications clearly precede the inflammation burst observed in human parturition and delivery and therefore might constitute an early event of parturition.

Materials and Methods

Placentas with their attached membranes were collected from: 1) healthy women with an uncomplicated pregnancy, undergoing a planned cesarean delivery at term; and 2) women with a pregnancy complicated by intrauterine growth restriction, preeclampsia, or chronic retroplacental hematoma, undergoing an emergency cesarean delivery for abnormalities in fetal heart rate between 28 and 35 wk of gestation (see Table I). The study of the human fetal membranes was approved by the local ethics committee (Comité de Protection des Personnes Ile de France XI, no. 11018, March 3, 2011). All participants provided written informed consent prior to inclusion in the study at the Department of Obstetrics, Port Royal Maternity, Cochin University Hospital, Paris, France.

Fetal membranes overlying the cervical os were marked during surgery with nontraumatic Duval grasping forceps, and the macroscopic area of swelling surrounding the forceps was retrieved. The ZAM and the ZIM were collected. Fetal membranes were separated from the placenta in the operating room under sterile conditions. Within 15 min of collection, biological samples were processed in the laboratory. Blood clots were removed by scratching the membranes and rinsing them in PBS. Choriodecidua was peeled away from the amnion. Pieces of choriodecidua (early preterm [EP], moderate preterm [MP], and term not labor [TNL] samples) were prepared as appropriate for RNA extraction and immunohistochemistry (IHC) experiments. TNLZAM and TNLZIM samples were processed for flow cytometry (FC) and electron microscopy (EM) analysis.

Total RNA was isolated using a Qiagen RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The purity and concentration of total RNA were first evaluated using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA), by measuring absorbance at 260 nm, and the samples were then analyzed on a Bioanalyzer system (Agilent Technologies, Santa Clara, CA) to verify their integrity. All samples with an RNA integrity number >6.5 were stored at −80°C until further processing.

Microarray analysis was conducted on 24 distinct samples divided into pools, with each pool containing three paired sets of choriodecidua samples. Gene expression was assessed by hybridizing the cRNAs to Agilent SurePrint G3 human GE 8 × 60K microarrays (Agilent Technologies) and assessing the fluorescence intensity for the individual oligonucleotide probes. Microarray raw data were imported into R v.2.15 and analyzed with the BioConductor limma package (19). Among the 62,956 probes present on the microarray, we included the 18,561 unique RefSeq reporters for further analysis. Genes with a nominal p value of ≤0.05 were considered to be differentially expressed. Genes showing >2-fold variation were further considered in the analysis. Hierarchical clustering and Gene Ontology (GO) terms analysis were performed using the MeV package (20) and the Genomatix pathway system (21–23) (release 2.4.0; Genomatix, Munich, Germany), respectively. Transcriptomic data reported in this study are archived in Gene Expression Omnibus database (accession nos. GSE50900 and GSE59686.

Quantitative RT-PCR

RNA was treated with DNase (Invitrogen/Life Technologies, St. Aubin, France) to remove any contaminating DNA. Four micrograms of total RNA was reverse transcribed using random primers and Moloney murine leukemia virus reverse transcriptase (Invitrogen), according to the manufacturer’s instructions. A negative control without RNA was included in each series of reverse transcription reactions. A set of genes, including three reference genes (internal controls), was analyzed by quantitative RT-PCR (RT-qPCR) using cDNA synthesized from each sample. Primers for RT-PCR analysis were chosen using Primer3 software, based on published sequences (available upon request). Primers were obtained from Eurogentec (Angers, France) and used at 10 nM in the PCR reaction. Quantitative PCR was carried out on a LightCycler 480, 96-well apparatus (Roche Diagnostics, Mannheim, Germany), with 160 ng of cDNA as a template. We used the amplification kit LightCycler 480 SYBR Green I Master (Roche), according to the manufacturer’s instructions. The efficiency of RT-PCR for all genes was estimated to be ≥90%. The abundance of each target gene was normalized to that of a set of internal controls, and a fold change was determined by the −2ΔΔCt formula. The set of internal controls included the geometric mean of three different reference genes, that is, succinate dehydrogenase complex subunit A, ribosomal protein large P0, and heterogeneous nuclear ribonucleoprotein A1. The results were analyzed by the LightCycler software using the three fit points method.

Transmission EM.

Fetal membranes were fixed in 4% paraformaldehyde/2% glutaraldehyde prepared in Sorensen buffer. After three washes in Sorensen buffer supplemented with 1% BSA, membranes were postfixed with 1% osmium tetroxide in 0.1 M phosphate buffer and then dehydrated by successive washing in 70, 90, and 100% ethanol. After being incubated for 10 min in a mixture of epoxy propane and epoxy resin (1:2), the samples were embedded in gelatin capsules with freshly prepared epoxy resin and polymerized at 60°C for 24 h. The samples were then mounted in Epon blocks and 90-nm semithin sections were obtained with an ultramicrotome (Reichert Ultracut S). The sections were stained with uranyl acetate and Reynold’s lead citrate and observed under a transmission electron microscope (Philips CM10).

Immunohistochemistry.

Tissues were harvested, fixed in 10% formalin, and embedded in paraffin. IHC was performed as described previously (26). Immunoreactivity was detected using the Novolink polymer detection system according to the manufacturer’s recommendations (Leica Microsystems, Nanterre, France). Color was developed using diaminobenzidine, and tissue sections were counterstained with Mayer’s hematoxylin solution (Merck Millipore, Guyancourt, France). Anti–HLA-F Ab, rabbit polyclonal 14670-1-AP (27) (Proteintech, Manchester, U.K.), and anti–HLA class 1 ABC, mouse clone EMR8-5 (28) (Abcam, Cambridge, U.K.), were diluted 1:100 in PBS/1% BSA/0.1% Triton X-100. The same combinations of Abs were previously used to detect alteration of HLA-F and HLA-I Ag expression in human esophageal squamous cell carcinoma (29). IHC signals were analyzed using National Institutes of Health ImageJ software to quantify the staining within the chorion trophoblastic layer in a ×20 field of view under a microscope equipped with a DC 300F camera (digital module R, IM 1000) (Leica Microsystems) by two independent investigators (J.D. and C.M.). Twenty images were randomly taken per sample and analyzed.

Statistical analysis

All data were analyzed by Prism 6 software (GraphPad Software, San Diego, CA). We used a Student t test for quantitative variables and a Pearson χ2 or Fisher exact test for qualitative variables, as appropriate. Nonparametric tests were used for non–normally distributed data. A p value <0.05 was considered to be significant.

Results

The choriodecidua overlying the cervix expresses a specific subset of genes prior to delivery at term

We compared the gene expression profile of choriodecidua overlying the cervix (ZAM) with that of regions located far from the cervix (ZIM), obtained from fetal membranes collected after cesarean section at 28–29 wk of gestation (EP samples), at 33–34 wk (MP samples), and at the term of gestation, prior to labor (TNL samples) (Fig. 1A, 1B, Table I). Principal component analysis indicated that the overall variability (>96%) in gene expression could be summarized by two major components: 1) the F1 (horizontal) axis, which groups all samples because of their common origin (choriodecidua), despite adverse maternal and fetal conditions associated with the preterm samples; and 2) the F2 (vertical) axis, which separates unequivocally TNLZAM samples from all other samples (Fig. 1C). Unsupervised hierarchical clustering of global gene expression identified two main clusters, separating again TNLZAM samples from all other samples (Fig. 1D).

The choriodecidua overlying the cervix at term presents a unique molecular signature. (A) The choriodecidua overlying the cervix (ZAM) and that located far from the cervix (ZIM) was sampled from intact membranes collected from pregnant women undergoing a cesarean section. (B) Samples were taken before labor between 28 and 29 wk of gestation, an EP stage (EP samples), between 33 and 34 wk of gestation, a MP stage (MP samples), and at term before labor (TNL samples). Transcriptomic analysis was performed on biological replicates of paired pooled samples. The number of women who were recruited in each group is indicated in the table. (C) Principal component analysis of gene expression. Each vector represents a pool of biological replicates. (D) Hierarchical clustering of the probes across ZAM and ZIM pools.

Induction of an early graft rejection-like program and subsequent inflammation

In TNL, 316 genes were significantly differentially expressed between ZAM and ZIM (variation ≥2-fold): 94 were upregulated in ZAM and 222 were downregulated (Fig. 2A). We compared these results with those obtained with a prior cohort of fetal membranes by Nhan-Chang et al. (30), who investigated gene expression in the choriodecidua at the site of rupture after spontaneous vaginal delivery at term (term in labor [TIL]). Among the 677 genes found deregulated after vaginal delivery (TIL) in the Nhan-Chang cohort, 124 were also deregulated in TNLZAM in our study (39.2% of 316; Pearson χ2 test, 906.1, p = 4.68 × 10−199). Many fewer genes were significantly deregulated in ZAM before term than after term: 84 genes were deregulated in moderate preterm ZAM (52 upregulated versus 32 downregulated genes), and only 17 genes were deregulated in extreme preterm samples (6 upregulated versus 11 downregulated genes). To validate these expression profiles, we performed RT-qPCR for a series of genes and found a significant correlation (p < 0.0001) between these and our microarray data (Supplemental Fig. 1).

A local graft rejection–like process occurs selectively at the site of subsequent membrane rupture. (A) Size of the gene lists. The gene expression arrays identified 17 genes differentially expressed between ZAM and ZIM in EP, 84 genes in MP, and 316 genes in TNL samples. Nhan-Chang et al. (30) described 677 genes that were differentially expressed at the site of rupture after vaginal delivery (TIL samples). (B) A Venn diagram was constructed to compare the four gene lists and to identify genes that were differentially expressed in ZAM according to term of pregnancy. (C) These groups of genes were analyzed for GO and sorted based on −log10 (p value). (D) A heat map representation of the genes related to inflammation that were differentially expressed either in both TNL and TIL or in TIL only, which shows that acute inflammation genes are deregulated in TIL but not TNL samples.

From the four (EP, MP, TNL, and TIL) gene lists (Fig. 2B), we mapped the chronological changes occurring in the choriodecidua overlying the cervix from 28 wk of gestation to rupture at term. GO analysis showed that ECM-related genes were significantly enriched after 33 wk of gestation (Fig. 2C). Among the 316 genes that were significantly differentially expressed in TNLZAM, most (n = 178) were only deregulated at that particular moment of pregnancy and had immune system–associated functions, most notably in the control of NK cells, T lymphocytes, lymphocyte activation, and graft rejection. Thus, the transcriptional activation of immunity-related genes was a distinctive feature of TNLZAM. Genes previously implicated in pregnancy or labor complications were significantly enriched among those that were uniquely upregulated after spontaneous delivery (n = 537). The GO term “Inflammation” described both the subset of genes that were commonly deregulated in “TNL and TIL,” and those that were deregulated “only in TIL.” A heat map representation of these inflammation-related genes showed that most were downregulated either in TNL or in TIL (Fig. 2D). Genes deregulated both in TNL and TIL and with a fold change >4-fold in TIL (27 genes) were mainly related to graft-versus-host disease (p = 3.6310−8) and ECM (1.98 × 10−7). Among the inflammation-related genes deregulated only in TIL, eight were overexpressed with a fold change >4-fold, including IL-6, TNF, and IL-8. This gene set was significantly enriched in genes associated with the inflammatory response (1.98 × 10−12), response to wounding (1.09 × 10−9), and acute phase response (5.00 × 10−9). We confirmed by RT-qPCR that acute inflammation-related genes, such as TNF and IL-6, were not upregulated in TNLZAM (Supplemental Fig. 1). Collectively, these observations illustrate that acute inflammation genes are preferentially expressed after labor and delivery.

Activation of NK cells and loss of M2-like macrophages in the choriodecidua overlying the cervix at full-term pregnancy precedes membrane rupture

We compared the composition of decidual immune cells in the samples obtained at full-term pregnancy before labor from ZAM and ZIM by FC. The proportions of CD45+ cells among the lymphocyte population were comparable between TNLZIM and TNLZAM (Supplemental Fig. 2 and recapitulative data in Table II). The percentages of lymphocytes CD45+CD3+ and of the subpopulations CD4+ and CD8+ T cells were also comparable between TNLZAM and TNLZIM. We evaluated the abundance of Tregs in the total CD4+ population and found that the proportions of expanded Tregs and naive Tregs (27) did not differ significantly between TNLZAM and TNLZIM (Supplemental Fig. 3A). Considering the NK cells, the proportion of CD3−CD56+NKp46+ cells significantly decreased ∼10% in TNLZAM versus TNLZIM whereas CD3−CD56+ cell percentages were similar between the two choriodecidual sites (Supplemental Fig. 2, Table II). Three subpopulations of CD56+NKp46+ dNK cells were discriminated using CD56 and CD16, an activation marker (Fig. 3A). A subpopulation of CD56brightCD16− cells accounted for almost 75% of the dNK cells in TNLZIM; they expressed high levels of the inhibitory receptor NKG2A, of the CD69 marker, and of NKG2D at their surface (Table II). The two other subpopulations, CD56dimCD16− and CD56dimCD16+, accounted for ∼15 and 10%, respectively, of dNK cells in TNLZIM; these subpopulations also present high, but to a lesser extent, expression of NKG2A, CD69, and NKG2D markers. The CD56dimCD16+ subset presented, in addition, high levels of the activating DNAM-1 receptor and of the chemotactic receptor CXCR3. Strikingly, the relative composition in these three subsets shifted in TNLZAM, with a significant decrease in the percentage of CD56bright NKs and a significant increase in the percentages of CD56dim CD16− and CD16+ NK cells, reaching >25% each of total dNKs. The expression levels of the activation marker HLA-DR was significantly higher in the three subsets of dNKs in TNLZAM, as well as the expression of the adhesion/homing molecule CD62L, whereas NKG2D levels decreased. Expression of CXCR3 also increased significantly in the CD56brightCD16− and CD56dimCD16− subsets. Overall, these data indicate that dNK cells are activated in TNLZAM, presenting novel repertoires of activating and chemotactic molecules.

NK cell and macrophage analyses in choriodecidual samples obtained at full-term pregnancy before labor from ZIM and ZAM. ZIM and ZAM cells were dissociated by collagenase treatment, and NK cells (A) and macrophages (B) were identified by FC. Depicted are the final gating strategies, the pie chart representation of the composition in cell subsets, and histograms showing the distinct cell subset percentages in matched TNLZIM and TNLZAM. Blue represents data from TNLZIM; red represents data from TNLZAM. Representative FC plots of independent experiments with dispersed choriodecidual cells from 8 to 10 women are shown. A paired nonparametric Wilcoxon test was applied. **p < 0.01.

Finally, we evaluated the proportions of macrophages between TNLZIM and TNLZAM (gating details are in Supplemental Fig. 4). The proportions of single cells in the macrophage area were comparable between TNLZIM and TNLZAM (43.07 ± 3.64 versus 41.39 ± 4.76, respectively; Supplemental Fig. 3B). The percentage of total CD14++ cells were significantly decreased in TNLZAM compared with TNLZIM (15.66 ± 2.16 versus 31.83 ± 2.59, respectively). Two subsets of CD14++ cells were distinguishable in this area (Supplemental Fig. 3B): 1) a subset of large cells, CD11c+, C16+, presenting high expression of CD206, the macrophage mannose receptor, characteristic of an M2-like subpopulation polarization; and 2) a subset of smaller cells, CD11c+, but mainly CD16− and CD206−. We then analyzed the proportions of CD14++CD11c+CD16+, M2-like, and CD14++CD11c+CD16− subsets (Fig. 3B). We found, in TNLZAM compared with TNLZIM, a significant decrease of the percentage of the M2-like cells (56.10 ± 4.52 versus 81.26 ± 1.64, respectively) and an increase of the CD14++CD11c+CD16− subset proportion (42.46 ± 4.78 versus 17.94 ± 1.71, respectively) within the CD14++ subpopulation. However, because the percentage of total CD14++ cells decreased in TNLZAM compared with TNLZIM, these percentage changes reflect, rather than an increase in the CD14++CD11c+CD16− population, a dramatic loss of M2-like cells in TNLZAM (see pie chart in Fig. 3B).

Immune activation at full-term pregnancy in choriodecidua overlying the cervix is associated with extraplacental trophoblast and ECM damage

We subsequently mapped the cell alterations in the samples obtained at full-term pregnancy before labor distinguishing ZAM from ZIM by performing EM analyses. TNLZIM contained many lymphocytes close to decidual cells and healthy trophoblasts as well as macrophages adherent to lymphocytes, lymphocytes with compacted chromatin, and resting NK cells (Fig. 4A). In contrast, TNLZAM contained complex cell aggregates made up of apoptotic trophoblasts, enlarged vacuolated macrophages, lymphocytes with unpacked chromatin, and vacuolated NK cells (Fig. 4B). Additionally, the ECM surrounding TNLZIM was dense and well organized, but that surrounding TNLZAM was diffuse and dispersed.

Exposure to different immunogenic HLA haplotypes at full-term pregnancy in choriodecidua overlying the cervix before labor

Because maternal lymphocyte activation may result from the presentation of fetal Ags, we examined the MHC status of fetal cells in the samples obtained at full-term pregnancy before labor from ZAM and ZIM. The HLA class Ia and Ib molecules, except HLA-F, are within the 2% most expressed genes detected by the microarrays (GSE50900). This observation reflects the mixed composition of the maternal cells and fetal cells in the choriodecidua. HLA-F and HLA-B mRNA expression was significantly lower in TNLZAM than in TNLZIM (log2 fold change of −1.17, p = 0.041 and log2 fold change of −1.1, p = 0.035, respectively). Regarding their cell surface expression, IHC analysis showed that immunostaining with an Ab that recognizes HLA-F protein (27) was present at the cell surface of trophoblasts in the chorionic layer in EP and MP samples (Fig. 5A), and abundance of the signal did not differ significantly according to location in ZIM or ZAM. In contrast, surface levels of HLA-F apparent staining in the chorionic layer were significantly lower (2.5-fold) at term in TNLZAM than in TNLZIM (p = 0.031). Conversely, classical polymorphic MHC class I, HLA-ABC protein staining with an Ab raised against the extracellular domain of HLA-A H chain (28) was low and unchanged in chorionic trophoblasts, as expected in EP and MP samples, but it was significantly higher in TNLZAM than in TNLZIM (3.51-fold; p = 0.016; Fig. 5B). Absence of HLA-F and HLA-ABC Ab cross-reactivities was checked by Western blot analysis (Supplemental Fig. 4). The HLA-F Ab interacted with a unique ∼48-kDa band in human choriodecidua trophoblastic cell extract (predicted HLA-F molecular mass of 45 kDa). The same Ab stained a faint band of the same apparent molecular mass in human choriocarcinoma JEG-3 cell lysate. In contrast, analysis of the same cell lysates with the HLA-ABC Ab reveals a different 36-kDa band (predicted molecular mass for HLA class I of 41 kDa). Abs directed against HLA-E or HLA-G stained also different bands, with 50 and 56 kDa molecular mass, respectively. Higher than predicted HLA-E and HLA-G molecular mass (45 and 40 kDa, respectively) observed in our assays possibly reflects glycosylation or other posttranslational modifications. Collectively, it can be concluded from the control experiments mentioned above that HLA-F and HLA-ABC Abs do not, as expected, recognize the same molecules. Our results are therefore consistent with a shift in HLA expression pattern occurring at term in TNLZAM (Fig. 6).

Disappearance of nonpolymorphic HLA-F molecules and de novo expression/presentation of classical highly polymorphic HLA-A, -B, and -C on chorionic trophoblasts in ZAM in choriodecidual samples obtained at full-term pregnancy before labor, but not in preterm membranes. (A and B) Immunohistochemistry analysis using anti–HLA-F and HLA-A, -B, and -C Abs. High magnification of fetal membrane sections collected from paired EPZIM and EPZAM samples (left panel) and paired TNLZIM and TNLZAM samples (right red panel). Bound Ab was detected with diaminobenzidine as a chromogen (brown deposits correspond to signal). The data are representative of paired TNLZIM and TNLZAM collected from six different women, as well as paired EPZIM and EPZAM and paired MPZIM and MPZAM collected from six different women. Inset, Representative images of control IHC performed with normal rabbit serum for HLA-F and with control IgG for HLA-ABC. Scale bars, 100 μm. Bottom, Quantification of staining within the chorionic layer before (EP) and at (TNL) term. Blue indicates data from ZIM; red indicates data from ZAM. A paired nonparametric Wilcoxon test was applied. *p < 0.05. Amn, amnion; Chor, chorion; Dec, decidua.

Summary of the cellular modifications that occur in ZAM at the end of pregnancy before the onset of labor, leading to a (semi) allograft rejection–like process. Within the zone of the choriodecidua overlying the cervix at the end of pregnancy, NK cells and macrophages change phenotype, becoming activated and phagocytic, respectively. This is associated with an increase in the expression of classical HLA-A, -B, and -C molecules and the disappearance, at the surface of chorionic trophoblasts, of the nonclassical HLA-F molecules that exhibit few polymorphisms.

Discussion

McLaren et al. (7) first described the unique morphological changes that occur after a full-term gestation before labor in a restricted area in fetal membranes located over the cervix in humans. These histological alterations comprise a marked disruption of connective tissue and the thinning down of the trophoblast and decidual layers. ECM remodeling the cellular layers are associated with a physical weakening of this zone (31), providing strong support to the idea that susceptibility to rupture is developmentally programmed. In this study, we show that decidual NK cells are activated locally in the choriodecidua overlying the cervix at full-term pregnancy, but not in earlier terms of gestation.

Through global gene expression and FC analysis, we demonstrate that decidual immune cells are specifically activated at this site. In particular, NK cells and M1-like macrophages become activated, and nonclassical MHC HLA-F molecules are replaced with classical, highly polymorphic, HLA-A, -B, and -C molecules at the surface of trophoblasts. We also analyzed the zone located over the cervix at two additional time points; between 28 and 29 wk and between 33 and 34 wk of gestation. For obvious ethical issues, the collection of preterm membranes in the absence of labor depends on a cesarean delivery. Our preterm samples were obtained from women, not in labor, who underwent emergency cesarean sections at early and midgestation for fetal distress; these women were hospitalized for daily monitoring because of diagnosed intrauterine growth restriction, maternal preeclampsia, and one case of retroplacental hematoma. Although these syndromes may be related to systemic or local inflammatory responses, preeclampsia and intrauterine growth restriction are not associated with an increased risk of spontaneous preterm delivery or premature rupture of membranes (32). Moreover, our principal component analysis of choriodecidua gene expression did not distinguish full-term ZIM from preterm samples. We also performed matched analyses, comparing the area overlying the cervix to a nearby area of the same patient, mitigating systemic condition impact on the analyses. This enabled us to determine the chronological changes occurring during ZAM induction and to relate this process to a unique immune signature at full-term pregnancy. At the EP stage, only 17 of the ∼24,000 human genes analyzed were differently expressed between ZAM and ZIM. Four weeks later into pregnancy, 84 genes, mainly related to ECM, were differentially expressed in the zone overlying the cervix. However, strikingly, at full-term gestation and before labor, >300 genes were differentially expressed in this area. In total, 280 of these genes were not deregulated in EPZAM or MPZAM and encode primarily immune markers and molecules involved in the graft rejection process.

Parturition is currently thought to involve the outbreak of intrauterine inflammation accompanied by the massive release of proinflammatory cytokines, such as IL-6 and TNF-α, uterotonins, for example, PGs PGE2 and PGF2α, and the influx of maternal leukocytes (3, 33). However, these observations are based on comparisons made between gestational tissues obtained after cesarean before labor and those obtained after vaginal delivery, without mention of specific regions within the uterus. Such studies reflect the end of the delivery process and provide limited insight into the temporal events triggering parturition and spontaneous labor. Here again, our comparison of matched tissues again smoothed out interindividual differences and strengthened our results. Remarkably, the comparison of our results with a list of genes differentially expressed between the site of rupture and an intact zone of the fetal membranes in women who delivered vaginally (30) converges to show that the expression of genes related to graft rejection, NK and lymphocytes, and leukocyte activation precedes the onset of spontaneous labor. Conversely, acute inflammation, illustrated by a 4-fold increase in IL-6, TNF, and PTGS2 expression, occurs only after labor onset. Therefore, we propose that inflammation does not trigger human parturition; instead, it may represent a downstream mechanism mainly involved in tissue remodeling and clearance of foreign material.

To examine how the transcriptional modifications described above relate to phenotypic and functional consequences, we analyzed the decidual leukocyte infiltrate in matched TNLZIM and TNLZAM samples. The proportion of CD45+ cells was similar in TNLZIM and TNLZIM, arguing against a global influx of leukocytes into the future site of rupture before labor. These findings are consistent with those of Osman et al. (34) who previously investigated leukocyte recruitment in different regions of the fetal membranes by IHC and failed to demonstrate an influx of CD45+ leukocytes at term before labor. Instead, we found dynamic changes in dNK (CD3−CD56+NKp46+) subpopulations before labor, with the TNLZAM containing a higher proportion of CD56dim CD16− and CD16+ cells than did the TNLZIM. CD56bright dNK cells are the predominant decidual cell population during early pregnancy, characterized by high expression of NKG2A and CD69 (35). The abundance of CD56bright NK cells in the decidua was recently reported to increase between the first and second trimesters (36). CD56bright NK cells produce large amounts of cytokines, whereas CD56dim NK cells are typically cytotoxic effectors. In early pregnancy, dNK cells are crucial for acceptance of the semiallogeneic fetus and for vascular transformation of the uterine spiral arteries (12, 13). Additionally, the presence of many CD56bright NK cells through midgestation creates an angiogenic and tolerogenic decidual microenvironment, which is necessary for harmonious fetal development. The reactivity of NK cells is controlled by several inhibitory and activating receptors at their surface and by their microenvironment. In the present study, we report that the activation of dNK cells is favored at the end of pregnancy. Indeed, the proportions of mature CD56dimCD16− and activated CD56dimCD16− double in ZAM, reaching more than half of the total NK cells in ZAM. The mean fluorescence intensity of HLA-DR and CD62L was significantly higher in TNLZAM than in TNLZIM for the three subsets of NK cells. Expression of CXCR3 is also higher in CD56bright and CD56dim CD16− cells, whereas it is already elevated in CD56dimCD16+ cells as well as in the expression of DNAM-1. An increase in HLA-DR presentation, which transmits Ag-related information to T cells, is also an index of activation, whereas DNAM-1 receptors enable functional interaction with macrophages (37). At term, we also observed a dramatic loss of CD206+ M2-like macrophages. EM images also revealed signs of global leukocyte activation, including cells showing structurally loose chromatin (indicative of active gene transcription) and a heavily vacuolated cytoplasm. Given that our transcriptomic data also suggested that T cell properties are fine-tuned at term in ZAM, we assessed the balance between conventional T cells and Tregs, the latter of which have an essential role in early pregnancy (38). Tregs, defined according to Miyara et al. (39), were scarce in fetal membranes and the Treg/conventional T cell balance was unchanged between ZAM and ZIM, consistent with data in mice suggesting that Tregs are dispensable in late pregnancy (40).

In early pregnancy, stromal cells and leukocyte populations within the decidua accommodate embryo implantation and the remodeling of the uterine environment that establishes the maternal–fetal interface. Extraplacental trophoblasts express a unique combination of MHC class I molecules, that is, HLA-G, HLA-E, HLA-F, and HLA-C at their surface, which help them to escape recognition by the maternal immune system. Among the HLA class 1b molecules, HLA-G was the first to be described at the surface of trophoblasts (41) and is one of the most characterized in early pregnancy, along with HLA-E. HLA-F, which has been less studied than HLA-G and HLA-E and presents even fewer polymorphisms, is strongly expressed at the surface of chorionic trophoblasts from midgestation throughout the last trimester of pregnancy (42). The function of HLA-F is not well understood, although it was recently described to be involved in evading immunosurveillance, tumor cell invasion, and metastasis (43). We observed that molecules, recognized by Abs raised against HLA-F, disappear from the trophoblast layer in TNLZAM at term before labor but continue to be expressed in TNLZIM. Moreover, TNLZAM chorionic trophoblasts start to express molecules that are recognized by Abs raised against HLA class Ia H chains at their surface. Although additional experiments are needed to address the specific identity of these molecules, these IHC data are consistent with our PCR and suggest a switch of HLA class I molecules at the surface of trophoblasts. Given the highly polymorphic nature of HLA-A, -B, and -C molecules and the semiallogenicity of trophoblasts, it is then likely that local exposure of paternal MHC Ags activates decidual lymphocytes near this area.

The cause underlying this local change in trophoblast MHC class 1 exposure is currently unknown. Temporal and topographical variations of mechanical properties within the lower segment of the uterus and the cervix are also well established; the fundus generates coordinated forceful uterine contractions while the contractile lower segment elongates over the presenting part and the cervix undergoes softening in late pregnancy (2). Mouse models of parturition demonstrated parturition-associated gene expression changes in the uterus, which are manifested prior to the acquisition of the contractile phenotype (8). Endocrine (progesterone withdrawal, corticotrophin releasing hormone), mechanical factors (uterine stretch), and/or fetal signals have been proposed to control the timing of parturition. Recently, Gao et al. (44) provided compelling evidence in mice that the fetus initiates parturition by signaling through at least two factors, surfactant protein A and platelet-activating factor, derived from the mature fetal lung. An increase in surfactant protein A and platelet-activating factor concentrations in the amniotic fluid activates fetal macrophages that migrate to the maternal uterus, inducing inflammation, progesterone withdrawal, and the activation of myometrial contractile genes (44). However, in humans, there is no evidence that fetal lung–derived factors may act as proinflammatory components or may promote the migration of fetal macrophages within maternal gestational tissues (45). The question of a maternal or fetal signal that will drive a polarized maturation of the trophoblasts in the area overlying the cervix remains open.

Because fetal membranes are ideally located to receive both maternal and fetal signals and to convey signals to the myometrium and cervix, a role of membranes in the initiation and propagation of labor has been previously proposed (1, 46). The rupture of membranes normally occurs after the initiation of myometrial contractions and cervical ripening. The presence of a region of structural weakness prior to labor might seem conflicting at first. However, the last critical sequences of membrane rupture are the nonelastic distension of the amnion and amnion rupture (47). Nhan-Chang et al. (30) reported no gene expression change in the amnion at the site of rupture compared with an intact region, inferring that amnion rupture is unlikely to be manifested some time prior to labor, unless displaying intrinsic physical weakness.

To conclude, at full-term pregnancy, we observed that chorion trophoblasts overlying the cervix change their phenotype and present haplotypes capable of activating maternal immune cells. We propose that this lymphocyte activation at the maternal–fetal interface highly focalized at the site of membrane rupture, followed by the local unleashing of a graft rejection–like program, represents an early event in human parturition (Fig. 6). These results have important medical implications in terms of delivery monitoring and control.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Florence Artiguebieille, Charlotte Leterme, Isabelle Avril, Sarah Guettouche, Victoria Buth, and Lauriane Kremer from the CICP 09-01 “Mère-Enfant” of Maternité Port Royal–Cochin for assistance in recruiting the women and collecting tissues samples, as well as the staff of the Operating Rooms of the Port Royal–Cochin Maternity. We are grateful to Shohreh Azimi of the Assistance Publique–Hôpitaux de Paris (Département de la Recherche Clinique et du Développement) and Laurence Lecomte-Raclet of the URC Paris Centre Necker–Cochin Assistance Publique–Hôpitaux de Paris for help in obtaining institutional legal authorizations to recruit pregnant women and to collect human tissues. We thank Juliette Nectoux for help with the quantitative PCR experiments, and the staff of the “Genomic,” “Histim,” EM, and “Cybio” platforms of the Cochin Institute for help with the transcriptomic, RT-qPCR, IHC, EM, and FC experiments, respectively. We also thank Emma Walton for editing the English of the manuscript.

Footnotes

This work was supported by Assistance Publique–Hôpitaux de Paris Grant CRC10134. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The transcriptomic data presented in this article have been submitted to the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo) under accession numbers GSE50900 and GSE59686.

. 2006. Separation of amnion from choriodecidua is an integral event to the rupture of normal term fetal membranes and constitutes a significant component of the work required.Am. J. Obstet. Gynecol.194: 211–217.